RNA is an important target for small molecule probes of function or lead therapeutics. Yet, very few RNAs have been exploited as such. Validated targets include ribosomal RNA,1, 2 which constitutes 80-90% of total cellular RNA,3-5 and riboswitches, that have known metabolite binders that can be mimicked to aid inhibitor design.6, 7 Compounds targeting the ribosome and riboswitches have been extremely useful probes to help understand RNA function. One major challenge in RNA chemical biology is how to exploit other RNAs in the transcriptome similarly. This is a considerable challenge given the low cellular abundance of these RNAs8 and the lack of lead small molecules.9
In an effort to exploit other potential RNA targets in the transcriptome with small molecules, we have developed a “bottom-up” strategy to design small molecules that bind an RNA of interest. That is, we define the small, discrete RNA secondary structural elements that are privileged for binding small molecules:10, 14 the interactions are then deposited into a database. The secondary structural elements in our selection studies are kept intentionally small such that they are likely components of larger cellular RNAs. The secondary structure of an RNA target is compared to our database of interactions, providing lead compounds. Leads can be optimized using various strategies including chemical similarity searching15, 16 and/or modular assembly.13, 17-22 One application of this strategy has been the rational design of bioactive small molecules that target the RNA that causes myotonic dystrophy-type 1 (DM1).15, 17, 18
DM1 is a presently incurable neuromuscular disease caused by a r(CUG) expansion, r(CUG)exp, in the 3′ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA.23, 24 The RNA folds into a hairpin structure that displays regularly repeating 1×1 nucleotide internal loops (5′CUG/3′GUC motifs; FIG. 1)25, 26 that are conformationally flexible.8, 22 The loops are high affinity binding sites for muscleblind-like 1 protein (MBNL1), and sequestration of MBNL1 causes its inactivation and subsequent dysregulation of alternative pre-mRNA splicing.28-31 Formation of the r(CUG)exp-MBNL1 complex causes various disease-associated defects including (FIG. 1): (i) pre-mRNA splicing defects;28, 32-34 (ii) formation of nuclear foci that consist of r(CUG)exp-protein complexes;35-37 ; and, (iii) translational defects of DMPK mRNA due to poor nucleocytoplasmic transport.38-40
Since the root cause of DM1 is r(CUG)exp, a variety of strategies have been employed to disrupt r(CUG)exp-MBNL1 complexes, thus releasing MBNL1 and restoring regulation of alternative splicing. Oligonucleotides that target r(CUG)exp improve DM1-associated defects upon injection into DM1 mouse models.33, 41, 42 Small molecules have also been developed that target r(CUG)exp including pentamidine, bis-benzimidazoles, naphthyl pyridines, and triazines.13, 15, 17-19, 43-46 The most potent are modularly assembled compounds that target the repeating nature of r(CUG)exp , binding multiple 5′CUG/3′GUC motifs simultaneously.13, 17-19 These compounds are composed of a modular assembly scaffold that displays multiple copies of an RNA-binding module on a single chain.13, 17-19